DESCRIPTION Protein farnesyltransferase (PFTase) catalyzes the farnesylation of Ras, and several other proteins, by farnesyl diphosphate (FPP). This modification is crucial for the localization and function of Ras, a protein implicated in up to 30% of human cancers. PFTase inhibition is a likely means of controlling Ras activity. Site-directed mutagenesis will be used to alter the ligands of the catalytically essential zinc co-factor such that the net charge of the zinc center will be changed. This will determine what influence the ligands have on the metal in substrate deprotonation and dissociation for nucleophilic attack on FPP. Mutants will be evaluated in regard to zinc binding, catalytic activity, pKa values for bound substrate thiol, and the ability to use peptide substrates with different nucleophiles. These experiments will aid in our chemical knowledge of the protein prenyltransferase enzymes and of the relatively new class of proteins that utilize a zinc-activated thiolate. The second major goal of this proposal is to use kinetic isotope effects to determine if cysteine farnesylation proceeds through an associative or a dissociative mechanism. Labeled FPP molecules will be synthesized in order to measure 2H and 3H alpha-secondary, 2H gamma-secondary, and 13C, 14C, and 18O primary kinetic isotope effects. Isotope effects and the structures of substrate-bound PFTases will be used to calculate a transition state structure which will be an excellent model for the rational design of PFTase-targeted anti-cancer drugs.